1. Field of the Invention
This invention relates generally to the art of studying the physical properties of microscopic particles carried in suspension and, more particularly, is concerned with improved structure for obtaining signals from hydrodynamically focused particles passing through a sensing aperture mounted in a module.
2. Description of the Prior Art
The type of particle analyzer, in which the teachings of the present invention are intended to be utilized, first was disclosed in U.S. Pat. No. 2,656,508 and operates on a principle often referred to as the principle of Wallace H. Coulter and improved upon as taught in U.S. Pat. No. 3,299,354 to Hogg. According to this principle, the passage of a microscopic particle suspended in a conducting liquid through a sensing aperture, having dimensions which are approximately ten times larger than that of the particle, causes a change in the impedance of the electrical path through the liquid contained in the aperture. The magnitude of this change is, under ideal conditions, approximately proportional to the volume of the particle. The particle analyzer typically includes a pair of electrodes positioned on either side of the sensing aperture. An electrical power source is coupled to the electrodes and a signal detecting circuit is connected across the electrodes, so that the signal detecting circuit will sense only impedance changes caused by the passage of a particle through the sensing aperture. These signals commonly are referred to as particle pulses and are fed from an amplifier to other electrical circuitry for the analysis of the pulse height and for counting the pulses.
Examples of particle analyzing devices having the structure and associated electrical circuitry described above can be found in products sold under the trademark COULTER COUNTER, which is a registered trademark of Coulter Electronics, Inc., Hialeah, Fla.
The pulse signal produced when a particle passes through the aperture depends not only on the particle size or volume, but also to a lesser extent on its shape, orientation, and its path near and through the aperture. For example, the amplitude-time history of the pulse signal for a particle moving near the aperture wall is different from that of a particle moving through the center of the aperture. To provide for better resolution of signals from closely sized particles, which are influenced by the above-described factors, several apparatuses have used "hydrodynamic focusing" techniques.
The most relevant apparatus using hydrodynamic focusing is shown in FIGS. 5 and 6 of U.S. Pat. No. 4,014,611 to Simpson et al., owned by the same assignee as that of the present invention. In this arrangement a nozzle is positioned between a bath of suspended particles and an aperture disk containing the sensing aperture, so as to define a sheath chamber therebetween. A passageway provides clean electrolyte to the sheath chamber. A liquid sample suspension of particles proceeds from the nozzle. The electrolyte in the sheath chamber forms a generally tubular sheath flow around the sample suspension. The hydrodynamic pressures of the sheath reduces the diameter of the sample suspension stream and centers it in a direction coaxial with the center axis of the sensing aperture. In this manner, the particles are hydrodynamically focused to travel in essentially the same path through the sensing aperture, thereby giving better signal resolution.
The above-described arrangement of U.S. Pat. No. 4,014,611 has several disadvantages when used in sophisticated commercial structures. First, the amount of sample suspension being processed cannot be simply and economically regulated or measured. More specifically, a regulator maintains a substantially constant pressure drop, and therefore a substantially constant flow, across the sensing aperture. Consequently, when there are variations in the amount of sheath liquid passing through the sensing aperture, there are forced variations in the flow of the sample suspension, since the total flow through the sensing aperture is held constant. Another problem is that even without such flow variations, to obtain a desired rate of flow of the sample suspension through the aperture, it is necessary to fix the rate of flow of the sheath liquid with respect to the rate of flow of sample suspension. Therefore, to obtain a constant non-fluctuating sample flow at a desired rate (i.e., fixed sheath to sample ratio) through the sensing aperture with this device, it is necessary in the prior art arrangement accurately and precisely to control both the sample suspension flow through the nozzle and the sheath flow, in addition to regulating the pressure drop across the sensing aperture. Second, the nozzle blocks the optical viewing of the sensing aperture, thereby preventing monitoring of the sensing aperture for possible clogging by debris. Third, since the upstream electrode is positioned on the upstream side of the sensing aperture, even the relatively large constrained path of the nozzle can introduce some signal noise, which causes a poorer signal to noise ratio. Fourth, the protruding end portion of the nozzle is unduly fragile and awkward for use in a commercial apparatus.
As discussed with U.S. Pat. No. 4,014,611, the hydraulic systems of the prior art for regulating and measuring the sample suspension through the sensing aperture require the control of the sample suspension flow and the sheath flow. The regulation of these flows require elaborate arrangements, such as the dual pistons of U.S. Pat. No. 4,001,678 to Berg. The Berg patent discloses another hydrodynamic focusing system using a liquid sheath around a sample suspension, wherein the pair of pistons force a quantity of liquid from a chamber downstream of a sensing aperture and also force a lesser quantity of liquid into a sheath chamber upstream of the sensing aperture, with the difference in the two quantities of liquid being made up by a quantity of sample suspension, which proceeds from a sample container, through a nozzle and into the sheath chamber.
U.S. Pat. No. 3,793,587 to Thom et al., assigned to the assignee of the present invention, discloses two dividing walls, each having an orifice and two sheaths for hydrodynamic focusing in successive chambers. The construction of this arrangement prevents the accurate measurement and regulation of sample suspension, which is sensitive to variation in the sheath flow and optical viewing of the sensing apertures.
Another hydrodynamic focusing arrangement is disclosed in an article entitled "Improved Resolution in Coulter Counting by Hydrodynamic Focusing", JOURNAL OF COLLOID AND INTERFACE SCIENCE, Vol. 26, pp. 175-182(1968). In this apparatus an elongated protrusion, mounted on the sample vessel, causes the suspension to be accelerated and thinned by a sheath of electrolyte prior to passing through the sensing aperture. Again, this arrangement has the inability simply to meter the sample volume and view the aperture as described with respect to the above-described devices.
U.S. Pat. No. 3,710,933 to Fulwyler et al., discloses a hydrodynamic focusing apparatus wherein the sample suspension is provided by a sample introduction tube and the liquid sheath is provided by a sheath tube positioned in surrounding, coaxial relationship to the sample introduction tube. A microscopic sensing aperture is positioned at the end of the sheath tube. In addition to the above described deficiencies, the tubes are very susceptible to breakage, due to the extremely small size.
U.S. Pat. No. 3,739,628 to Karuhn et al. discloses a flow straightener means in the form of a flow directional collar. However, there is no sheath for surrounding the sample suspension and the collar merely reduces flow turbulence, while directing particles along a path somewhat coaxial with the center axis of the sensing aperture. Hence, there is no hydrodynamic focusing using a liquid sheath.
U.S. Pat. Nos. 2,656,508 to Coulter, 3,299,354 to Hogg, and 4,014,611 to Simpson et al. are incorporated herein as a part hereof by specific referece.